As is known in the art, meningitis may be broadly characterized as aseptic meningitis or bacterial meningitis. Aseptic meningitis may refer to two different but related forms of meningitis. Firstly, it may be referring to an acute febrile illness with meningeal irritation and cerebro-spinal fluid (CSF) pleocytosis, but with normal glucose contents and on culture of the CSF no bacterial isolation is obtained. The second form of aseptic meningitis can be characterized by CSF pleocytosis, normal glucose and the absence of organism involvement. This can include not only viral but other infections as well as non-infectious causes. When viral infections of the central nervous system are invovled, these infections may take three different forms. In the first form, there may be no symptoms and only fever and malaise, but the CSF may be abnormal with a lymphocytic pleocytosis. In a second form, the symptoms may be confined to meningeal manifestations, such as fever, headache, vomiting, stiff neck and back and the like. In the third form, referred to as encephalitis, the meningeal symptoms, noted above, may be accompanied by cerebral disorders including alterations of consciousness, personality changes and the like.
Bacterial meningitis may be caused by one or more of several known bacteria. Among the generally accepted bacteria are the Gram-negative bacteria Neisseria meningitidis, Haemophilus influenzae, Escherichia coli, Salmonella enteridis (or group B), Proteus species (indole positive and negative) and Pseudomonas arugeniosa, and the Gram-positive bacteria, Diplococcus pneumoniae, Strepococcus pyogenes, and Straphylococcus aureus. In bacterial meningitis, clinical indications show brain and meninges involvement. Some of the clinical signs can include fever, headache, stiff neck and vomiting.
Thus, from a clinical observation, it is difficult to tell whether aseptic meningitis or bacterial meningitis is involved, since the clinical symptoms of the different forms of meningitis can be, at least, overlapping in their manifestations. However, the proper treatment for the forms of meningitis vary significantly, and a rapid and positive diagnosis of the form of meningitis is extremely important in the treatment of meningitis. If the meningitis is diagnosed as bacterial meningitis, the more usual therapy involves administration of antibiotics, of specific types, and such administration should be given as promptly as possible once the diagnosis has been established. The prompt commencement of the thereapy is fundamental to the more successful treatment of bacterial meningitis.
Accordingly, when clinical symptoms of meningitis are observed, the first diagnostic procedure is that for distinguishing between aseptic meningitis and bacterial meningitis. Unfortunately, however, usually practiced laboratory means of differentiation between bacterial meningitis and aseptic meningitis may be inadequate or even misleading, particularly in distinguishing between bacterial meningitis and viral meningitis. As can be easily appreciated, the ability to make such distinctions is further significantly complicated when antibiotics have been administered to the patient prior to the testing for meningitis, since the antibiotics can substantially attenuate the bacterial infection and/or cause modification of the normal clinical symptoms thereof. It has been found, for example, that the administration of antibiotic thereapy prior to testing CSF for meningitis can cause a decrease of about 25% in the positive results of the CSF culture test and can cause a decrease of about 15% in the positive results of the Gram stain test (in this regard, see Lewis, E. B., "Partially Treated Meningitis," Am. J. Dis. Child, 128:145-147, 1974). These tests, therefore, can be totally inadequate to distinguish between aseptic and bacterial meningitis or at least misleading when there has been a prior administration of antibiotics.
It has also been found that tests for CSF pleocytosis, sugar and protein content can be confusing, even when no antibiotic pre-treatment has been given. For example, it has been found that approximately 10% of culture-proved cases of acute bacterial meningitis will exhibit low white cell counts with a predominance of lymphocytes in the CSF. Such findings are more normally associated with the classical characteristics of aseptic meningitis rather than bacterial meningitis. Similar misleading test results have been demonstrated in patients with Haemophilus influenzae meningitis, Neisseria meningitides and Diplococcus pneumoniae meningitis.
Conversely, cases of aseptic meningitis having high pleocytosis, which is more suggestive of a bacterial etiology, may be encountered. This is particularly true with meningitis caused by enteroviruses. Early in the course of Coxsackie and Echo virus meningitis, CSF cell counts may be well over 500 or even 1,000 white cells per cubic milliliter with a predominance of granulocytes. An analysis of cases of patients suffering from Echovirus (type 4) meningitis revealed that 25% of patients had a pleocytosis above 500 white cells per cubic milliliter and 70% had a predominance of granulocytes on the initial tap. Thus, it may be concluded the neither the degree nor the character of CSF pleocytosis is always helpful diagnostically, where the enteroviruses are involved, because of the overlap in the range of values between the "classical" aseptic and bacterial meningitis findings.
Similarly, a CSF sugar content study showed that CSF sugar determinations were normal in 35% of culture-proved cases of bacterial meningitis. Thus, CSF sugar values often lacked reliability as a diagnostic parameter.
In view of the above, the art has had a long-felt need for a more accurate test for distinguishing between aseptic and bacterial meningitis. An important advance in this regard was with the discovery of clot formation of amebocyte lysate from the Limulus polyhemus (horseshoe crab) in the presence of minute amounts of endotoxin produced by Gram-negative bacteria (see Leven, J., and Bang, F., "The Role of Endotoxin in the Extracellular Coagulation of Limulus Blood," Johns Hopkins Med. J., 115:265-274, 1965). This has led to proposals for using Limulus lysate in detection of Gram-negative bacteria (see Jorgensen, J., et al, "Rapid Detection of Gram-Negative Bacteriuria By Use of the Limulus Endoxtoxin Assay," Applied Microbiology, Vol. 26, No. 1, (1973) p. 38-42 and Jorgensen, J., Smith, R., "Rapid Detection of Contaminated intravenous Fluids Using the Limulus In Vitro Endotoxin Assay, " Applied Microbiology, Vol. 26, No. 4, (1973), p. 521-524). It has also been proposed to use the so-called Limulus lysate test for detection of bacterial meningitis. In one study, positive Limulus lysate assays were obtained on all initial CSF specimens from 38 patients with culture-proved Gram-negative bacterial meningitis. Of these 38 cases, 29 were due to H. influenzae and 7 to N. meningitidis. No positive Limulus lysate tests were noted in patients with Gram-positive bacterial meningitis, Tuberculosis meningitis, aseptic meningitis, or patients without meningitis. Thus, it was concluded that the Limulus lysate assay was a sensitive test for the diagnosis of untreated Gram-negative bacterial meningitis (see Nachum, R., Lipsey, A., and Siegel, S. E., "Rapid Detection of Gram-Negative Bacterial Meningitis by the Limulus Lysate Test, " New England J. Med., 289:931-934, 1973). It should be noted, however, that the reliability of testing Gram-negative bacteria by the Limulus lysate test is not without controversy and the accuracy thereof has been questioned (see Martinez, G. L., Quintiliani, R., and Tilton, R., "Clinical Experience on the Detection of Endotoxinemia With the Limulus Test," J. Infect. Dis., 127:102-105, 1973; Stumacher, R., Kovnat, M., and McCabe, W., "Limitation of the Usefulness of the Limulus Assay for Endotoxin," New England J. Med., 288:1261-1264, 1973; and Feldman, S., and Pearson, T. A., "The Limulus Test and Gram-Negative Bacillary Sepsis," Am. J. Dis. Child, 128:172-174, 1974). Nevertheless, it is believed that the Limulus lysate test is the best overall test to distinguish between bacterial meningitis and aseptic meningitis.
The test does suffer, however, from serious difficulties. As can be appreciated from the foregoing, it is of primary importance that the results of any test for distinguishing from aseptic meningitis and bacterial meningitis be made available to the clinician as soon as possible. Significant delays in obtaining the results of such tests can seriously undermine the effectiveness of thereapy instituted after those results are known. The conventional manner of carrying out the Limulus lysate test is described by Nachum et al, cited above. Basically, equal amounts of CSF and lysate are added to pyrogen-free glass culture tubes and incubated in a 37.degree. C water bath for 4 hours. Tubes are observed at 15-minute intervals during the initial incubation period. Tests still negative after the initial 4-hour incubation period are further incubated at room temperature for an additional 20 hours. A definite increase in viscosity and turbidity usually, but not necessarily, progressing to adherent granules or solid gel is interpreted as a positive test. Negative assays remain clear or contain slight flocculent precipitates suspended in clear fluid media. A scale of positive reaction is: negative-clear of slightly flocculated; +-- increased turbidity and viscosity; ++-- adherent granules; and +++-- solid gel. Thus, the test not only requires laboratory equipment and extended time periods, but the subjective determination of a positive or negative result requires an experienced laboratory technician. This substantially decreases the usefulness of the Limulus lysate test in differentiating between aseptic and bacterial meningitis. It would therefore be of significant advantage in the art to provide a modified Limulus lysate test which can be rapidly carried out with simple equipment, essentially at the bedside, and which does not require an experienced clinician to accurately interpret the test. As can be easily appreciated, experienced clinicians to accurately interpret the conventional test are not readily available to most practicians and an incorrect interpretation of a Limulus lysate test could cause most serious consequences in subsequent treatment for the meningitis.